Compulsive drug-seeking and drug-taking behaviors are hallmarks of drug-addiction and propensity to relapse is a significant problem. Cocaine-addiction is a human disease, in particular, a multifaceted mental disorder involving many neuro-adaptations and enduring changes in the neurobiology of addicted brain reflect in altered neurochemistry and behavior. Despite the identification of several putative pharmacological targets, there are no effective FDA approved pharmacotherapies for the treatment of cocaine addiction or relapse. Therefore, further understanding of addiction neurobiology is necessary to develop effective treatment strategies. Monoamine transporters are critical molecular targets that mediate abuse-related effects of psychostimulants. Our long-standing investigations lead us to a new line of thinking that monoamine transporters, DAT, NET and SERT are regulated neuronal targets of cocaine, dictated by signaling architecture of synapses associated with cocaine-addiction. Our studies have demonstrated that T30-dependent NET regulation plays a critical role in cocaine regulation of synaptic NE signaling, and interventions of this molecular event attenuate cocaine reward (PMC3121486; PMID: 25724654). Our recent preliminary data from in vivo and in vitro studies show that brain nuclei specific manipulation of NET-T30 phosphorylation can attenuate cocaine-reinforcing behaviors and that NET-T30E mutation mimics cocaine-exposed-like phenotype, the NET upregulation. However, the neurobiological consequences of in vivo NET-T30 phosphorylation are unclear, and studies using intact animal models are crucial to a clear understanding of the role of in vivo NET-T30 phosphorylation (the molecular mechanism) in cocaine reinforcing behaviors. As a continued effort to address this, we have developed prototypical mutant mouse models mimicking and lacking NET-T30 phosphorylation. In this R21 exploratory application, we propose to test an overarching hypothesis that while the NET-T30 phospho-mimicking NET- T30E mice exhibit gain of function and cocaine-exposed like behavioral phenotypes, the phospho-lacking NET- T30A mice exhibit normal transport function and insensitivity to cocaine-mediated effects (loss of cocaine induced transport upregulation and behaviors). Two specific aims will test this hypothesis. Aim 1 will study the effects of acute and chronic cocaine on animal behavior along with parallel analysis of NET functional expression and phosphorylation in NET-T30E mice. Aim 2 will Study the effects of acute and chronic cocaine on animal behavior along with parallel analysis of NET functional expression and phosphorylation in NET-T30A mice. Aims 1 and 2 will implement locomotor activation and sensitization as well as conditioned place preference (CPP) paradigms to study the behavioral response of NET-T30E and NET-T30A mice to cocaine. These NET-T30E and T30A mutant mouse models will be powerful biomolecular tools not only for basic neurobiological understanding of a fundamental problem, but also for directing our focus on intervening the underlying molecular mechanisms of cocaine actions.